Genome-Wide Identification and Evolutionary and Mutational Analysis of the Bos taurus Pax Gene Family

Bos taurus is known for its tolerance of coarse grains, adaptability, high temperature, humidity, and disease resistance. Primarily, cattle are raised for their meat and milk, and pinpointing genes associated with traits relevant to meat production can enhance their overall productivity. The aim of this study was to identify the genome, analyze the evolution, and explore the function of the Pax gene family in B. taurus to provide a new molecular target for breeding in meat-quality-trait cattle. In this study, 44 Pax genes were identified from the genome database of five species using bioinformatics technology, indicating that the genetic relationships of bovids were similar. The Pax3 and Pax7 protein sequences of the five animals were highly consistent. In general, the Pax gene of the buffalo corresponds to the domestic cattle. In summary, there are differences in affinity between the Pax family genes of buffalo and domestic cattle in the Pax1/9, Pax2/5/8, Pax3/7, and Pax4/6 subfamilies. We believe that Pax1/9 has an effect on the growth traits of buffalo and domestic cattle. The Pax3/7 gene is conserved in the evolution of buffalo and domestic animals and may be a key gene regulating the growth of B. taurus. The Pax2/5/8 subfamily affects coat color, reproductive performance, and milk production performance in cattle. The Pax4/6 subfamily had an effect on the milk fat percentage of B. taurus. The results provide a theoretical basis for understanding the evolutionary, structural, and functional characteristics of the Pax family members of B. taurus and for molecular genetics and the breeding of meat-production B. taurus species.


Introduction
Domestic cattle belong to the subfamily Bovidae of the even-toed ungulaceae and were one of the earliest-domesticated domestic animals in the course of evolution.The consensus on the origin of domestic cattle is that Bos primigenius, which was widespread in Eurasia in prehistoric times, is their ancestor.Bos primigenius is divided into three continental subspecies, B.p. primigenius, B.p. Nomadicus, and B.p. opisthonomus, according to the shapes and sizes of their horns.The family consists of two species, the Taurine and the Zebuine.Up to now, a large number of studies have shown that the common Auroprotozoa and the tuberoprotozoa have undergone multiple independent domestication events.The ancestors of Zebu cattle were domesticated in the Indus Valley about 8000 years ago [1].Common cattle were domesticated in Southwest Asia (present-day Turkey) about 10,000 years ago.The earliest archaeological evidence for the domestication of auropodial cattle is 10,750-10,250 years old [2].In recent years, the availability of high-quality reference genomes and moderately dense genotyping marker sets has stimulated a series of genome-wide studies on plant diversity, evolutionary history, production traits, and functional elements.As research advances and molecular knowledge continues to be integrated into breeding programs, the global domestic cattle population will gradually improve and strengthen in terms of yield, environmental adaptability, and disease resistance [3].
With the advent of modern molecular biology techniques, paired box (Pax) proteins were identified by researchers as being able to regulate gene transcription during biological development [4].Regarding the Drosophila pared (prd) gene, it was discovered that both the Drosophila prd and gooseberry (gsb) genes contain a conserved pared domain of 128 amino acids [5].These groundbreaking discoveries inspired us to understand the highly conserved transcription factors in the Pax family.In vertebrates, the Pax gene family is divided into four subfamilies, Pax1/9, Pax2/5/8, Pax3/7, and Pax4/6, based on the different structural features of the genes [6].Members of the Pax gene family are highly conserved throughout evolution and play critical roles in the organism, including the ability to coordinate the development of tissues and organs and to maintain cellular properties [7,8].
Members of the Pax gene family also play important roles in mammals, including the formation of the nervous system and the development of tissues and organs [9].Previous studies of the Pax gene family have not compared different species or explored the evolutionary relationships between gene families in different species.While genomic studies of species have historically been laborious and distant, gene family analysis has become an emerging trend.As large numbers of species genomes have been measured and data released, the identification of gene family members from species genomic data using specific structural models has become increasingly common [10].However, there is currently no comprehensive genome-wide study of the Pax gene family in Bos taurus that is being conducted by researchers.The purpose of this study was to identify the members of the Pax gene family in B. taurus and characterize their physicochemical properties.The joint analysis of Pax genes from related species was conducted to assess the functionality of Pax gene family members in B. taurus.
To identify potential Pax genes in these domestic animal species, using Pfam database (https://www.ebi.ac.uk/interpro/entry/pfam/) (assessed on 17 May 2024), a conservative HMG Pax structure domain protein sequence of the Hidden Markov model (Hidden Markov model, HMM) map was used.The model number was PF00292.The hmmsearch tool was used to search the domain sequence of the target species, and the E value was set to 1.2 × 10 −28 .The ClustalW tool was used to conduct multiple-sequence comparison of the results, and the hmm model was established again with the comparison results.Finally, the domain was searched again according to the newly established model.Genes with E-value < 0.01 were selected as members of Pax gene family.These were identified on the sequence of gene family members submitted to NCBI BatchCD-Search (https://www.ncbi.nlm.nih.gov/cdd)(assessed on 17 May 2024) for structural domain analysis and blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi)(assessed on 17 May 2024), confirming Pax gene structure domain after complete and accurate evaluation and subsequent analysis process.

Multiple-Sequence Alignment and Phylogenetic Analysis of Pax Gene Family
The ClustalW function in MEGA11 v11.0.13 software was used to compare Pax gene family sequences of domestic cattle, Zebu cattle, buffalo, pig, and sheep with default parameters, and the phylogenetic evolutionary tree of related species was constructed by adjacency method (NJ) and bootstrap setting 1000 times.And we imported the results to iTOLv6 (https://itol.embl.de/)(assessed on 17 May 2024) to visualize the results.

Sequence Analysis of Pax Gene in B. taurus
After obtaining Pax gene family sequences, we used ClustalW in MEGA11 to compare Pax sequences of domestic cattle and make NJ evolutionary tree.In addition, we uploaded the domain data of the Pax gene family to MEME (Multiple-Expectation Maximization for Motif Elicitation) website (https://meme-suite.org/meme/) (assessed on 18 May 2024) to obtain the motif characteristics of the family.The minimum and maximum widths of the motifs were set to 6 and 50, respectively, and the number of search motifs was set to 10.The exon-intron pattern of Pax gene was obtained by analyzing the structure of Pax gene using GSDS website (http://gsds.gao-lab.org/)(assessed on 18 May 2024).In order to explore the structural characteristics of the protein encoded by the Pax gene family in domestic cattle, MEGA11 ClustalW function was used to perform multiplesequence alignment of Pax protein sequences of domestic cattle, and the comparison results were imported into GeneDoc v2.7 software for analysis and visualization.

Protein Three-Dimensional Structure and Interaction Network of Pax Gene
In order to explore the three-dimensional protein structure of Pax gene, we imported the amino acid sequence of Pax gene into SWISS-MODEL website to predict it.We selected the template with high coverage and similarity in the prediction results.We selected a template with a large GMQE value and a QMEAN close to 0. After saving, we built up 3D model file and PDB file and used SAVES (https://saves.mbi.ucla.edu/)(assessed on 22 May 2024) site in the model analysis to judge the PDB file availability of threedimensional structure.
The protein interaction network of Pax gene was constructed using the online website STRING (https://cn.string-db.org/)(assessed on 22 May 2024) to explore the interactions among the proteins encoded by the Pax gene family and the results were imported into the software for beautification.

Systematic Evolution of the Pax Gene Family
Using the hmmer tool, nine Pax genes were found in the whole-genome data of domestic cattle, the Zebu, buffalo, and sheep, and eight Pax family members except Pax8 were found in pigs, with a total of 44 genes.According to the evolutionary relationships, these genes can be divided into four subfamilies, namely the Pax3/7 subfamily, Pax4/6 subfamily, Pax1/9 subfamily, and Pax2/5/8 subfamily.The selective evolutionary analysis of the five animals showed that domestic cattle were more homologous to buffalo and the Zebu than other domestic animals most of the time (Figure 1).
The protein interaction network of Pax gene was constructed using the online website STRING (https://cn.string-db.org/)(assessed on 22 May 2024) to explore the interactions among the proteins encoded by the Pax gene family and the results were imported into the software for beautification.

Systematic Evolution of the Pax Gene Family
Using the hmmer tool, nine Pax genes were found in the whole-genome data of domestic cattle, the Zebu, buffalo, and sheep, and eight Pax family members except Pax8 were found in pigs, with a total of 44 genes.According to the evolutionary relationships, these genes can be divided into four subfamilies, namely the Pax3/7 subfamily, Pax4/6 subfamily, Pax1/9 subfamily, and Pax2/5/8 subfamily.The selective evolutionary analysis of the five animals showed that domestic cattle were more homologous to buffalo and the Zebu than other domestic animals most of the time (Figure 1).

Gene Structure Characterization of Pax Gene Family in B. taurus
We analyzed the structure of the Pax gene family in domestic cattle.The results included the gene structure (Figure 2), phylogenetic tree (Figure 3A), motif (Figure 3B), conserved domain distribution (Figure 3C), and differentially conserved motifs in B. taurus's Pax gene family (Table 1).Gene structure analysis found that the number of exons and introns in each Pax gene was fixed, Pax9 had the least exons (four), and Pax2 had the most exons (thirteen).The number of UTRs ranged from one to five (Figure 2).A total of 10 motifs were identified from the Pax gene family of domestic cattle.The motifs of the Pax gene family ranged from 14 to 94 in length, and motif1, motif5, and motif7 all had Genes 2024, 15, 897 5 of 14 high amino acid quantities.Motif1 and motif2 are present in every gene family member.Through the study of motifs, we found that in the same gene subfamily, the number of motifs is often consistent.For example, the Pax3/7 subfamily has a relatively large number of motifs while the Pax1/9 subfamily has a relatively small number of motifs (Figure 3B).All Pax genes contain Pax domains; the homeodomain superfamily is found only in the Pax2 gene and the AKR_SF superfamily is found only in Pax4.The species and quantity distributions of conserved domains of genes within the same subfamily are also very similar (Figure 3C).
We analyzed the structure of the Pax gene family in domestic cattle.The results included the gene structure (Figure 2), phylogenetic tree (Figure 3A), motif (Figure 3B), conserved domain distribution (Figure 3C), and differentially conserved motifs in B. taurus's Pax gene family (Table 1).Gene structure analysis found that the number of exons and introns in each Pax gene was fixed, Pax9 had the least exons (four), and Pax2 had the most exons (thirteen).The number of UTRs ranged from one to five (Figure 2).A total of 10 motifs were identified from the Pax gene family of domestic cattle.The motifs of the Pax gene family ranged from 14 to 94 in length, and motif1, motif5, and motif7 all had high amino acid quantities.Motif1 and motif2 are present in every gene family member.Through the study of motifs, we found that in the same gene subfamily, the number of motifs is often consistent.For example, the Pax3/7 subfamily has a relatively large number of motifs while the Pax1/9 subfamily has a relatively small number of motifs (Figure 3B).All Pax genes contain Pax domains; the homeodomain superfamily is found only in the Pax2 gene and the AKR_SF superfamily is found only in Pax4.The species and quantity distributions of conserved domains of genes within the same subfamily are also very similar (Figure 3C).We used the WoLF PSORT website for the subcellular localization of the Pax gene family members and the results showed that all members of the Pax gene family were located in the nucleus (Table 2).

Multi-Sequence Alignment of Pax Protein in B. taurus
The ClustalW function of MEGA11 was applied to compare the protein sequence of the Pax gene in domestic cattle and the results were imported into GeneDoc to produce the following results (Figure 6).The red area in the figure below indicates that the comparison rate is 100%, the orange area has a comparison rate of 70% to 99%, the yellow area has a comparison rate of 50% to 69%, and the colorless area is less than 50%.The comparison results show that nine Pax protein sequences had high consistency in the 100-220

Multi-Sequence Alignment of Pax Protein in B. taurus
The ClustalW function of MEGA11 was applied to compare the protein sequence of the Pax gene in domestic cattle and the results were imported into GeneDoc to produce the following results (Figure 6).The red area in the figure below indicates that the comparison rate is 100%, the orange area has a comparison rate of 70% to 99%, the yellow area has a comparison rate of 50% to 69%, and the colorless area is less than 50%.The comparison results show that nine Pax protein sequences had high consistency in the 100-220 region and were highly conserved in this region.It was speculated that the conserved domain of the Pax family was located in this region, which was related to the function of the Pax protein.

Three-Dimensional Structure of Pax Protein
The SWISS-MODEL website was used to predict and select the three-dimensional structure of the Pax protein, which was evaluated by the website of SAVES for usability, as shown in the figure below (Figure 7).The three-dimensional structure of the protein is The SWISS-MODEL website was used to predict and select the three-dimensional structure of the Pax protein, which was evaluated by the website of SAVES for usability, as shown in the figure below (Figure 7).The three-dimensional structure of the protein is composed of a motif and domain.The activity and function of proteins are not only determined by the primary structure of protein molecules but also closely related to their unique spatial structures.The wrong spatial structures in proteins can lead to reduced function or even the inactivation of proteins, which can also lead to a range of diseases.For example, mad cow disease is caused by the aggregation of certain proteins after the misfolding, forming amyloid fiber precipitation against proteolytic enzymes, resulting in toxicity and disease.
Genes 2024, 15, x FOR PEER REVIEW 10 of 15 composed of a motif and domain.The activity and function of proteins are not only determined by the primary structure of protein molecules but also closely related to their unique spatial structures.The wrong spatial structures in proteins can lead to reduced function or even the inactivation of proteins, which can also lead to a range of diseases.For example, mad cow disease is caused by the aggregation of certain proteins after the misfolding, forming amyloid fiber precipitation against proteolytic enzymes, resulting in toxicity and disease.

Protein Interaction Network of Pax Gene
The protein interaction network of Pax genes was constructed using the online website STRING and we imported the results into cytoscape software for beautification (Figure 8).The results showed that only the Pax3 and Pax7 genes had direct interaction in the Pax gene family and the interactions between other Pax genes needed to be completed through the transfer of genes outside the family.

Protein Interaction Network of Pax Gene
The protein interaction network of Pax genes was constructed using the online website STRING and we imported the results into cytoscape software for beautification (Figure 8).The results showed that only the Pax3 and Pax7 genes had direct interaction in the Pax gene family and the interactions between other Pax genes needed to be completed through the transfer of genes outside the family.

Discussion
B. taurus is one of the world's most important livestock, a cornerstone of the world's livestock industry, providing beef as an important source of protein for humans.B. taurus was one of the first domesticated animals to have spread around the world with human migration and trade, allowing it to genetically adapt to different climatic conditions in different regions [3,11].B. taurus provides traction for farmers, improves agricultural production efficiency, and made an important contribution to the development of farming civilization.The Pax gene family is closely related to the growth traits of B. taurus [12].The study of the Pax gene family can further increase beef yield to meet the increasing beef consumption demand of people.The evolution and gene structure of the Pax gene family were studied to provide a theoretical basis for breeding B. taurus with better meat quality traits.
Our study identified 44 members of the Pax gene family in five species by bioinformatic techniques.In this study, a phylogenetic analysis of Pax genes from species was performed to explore the differences in affinities, divergences, and motifs.Phylogenetic analyses of Pax family genes provided an in-depth understanding of the evolution of the Pax family genes.Neighbor-joining tree analysis showed that Pax family genes could be divided into four taxa, and the affinities of Pax genes differed among species of different genera within each taxon.The results showed that B. taurus genes have diverged during the evolution of the species and that not all genes are related in the same way.The affinities of Pax genes varied within the Pax1/9, Pax2/5/8, Pax3/7, and Pax4/6 subfamilies among buffalo and domestic cattle.
Pax1 is essential for the differentiation of the thymus, vertebrae, and cartilage and the

Discussion
B. taurus is one of the world's most important livestock, a cornerstone of the world's livestock industry, providing beef as an important source of protein for humans.B. taurus was one of the first domesticated animals to have spread around the world with human migration and trade, allowing it to genetically adapt to different climatic conditions in different regions [3,11].B. taurus provides traction for farmers, improves agricultural production efficiency, and made an important contribution to the development of farming civilization.The Pax gene family is closely related to the growth traits of B. taurus [12].The study of the Pax gene family can further increase beef yield to meet the increasing beef consumption demand of people.The evolution and gene structure of the Pax gene family were studied to provide a theoretical basis for breeding B. taurus with better meat quality traits.
Our study identified 44 members of the Pax gene family in five species by bioinformatic techniques.In this study, a phylogenetic analysis of Pax genes from species was performed to explore the differences in affinities, divergences, and motifs.Phylogenetic analyses of Pax family genes provided an in-depth understanding of the evolution of the Pax family genes.Neighbor-joining tree analysis showed that Pax family genes could be divided into four taxa, and the affinities of Pax genes differed among species of different genera within each taxon.The results showed that B. taurus genes have diverged during the evolution of the species and that not all genes are related in the same way.The affinities of Pax genes varied within the Pax1/9, Pax2/5/8, Pax3/7, and Pax4/6 subfamilies among buffalo and domestic cattle.
Pax1 is essential for the differentiation of the thymus, vertebrae, and cartilage and the maturation of chondrocytes during embryonic development [13].In African clawed toad embryos, Pax1 is detected early in somitogenesis and expressed at increased levels in the osteogenesis and endodermal pharyngeal sacs [14].Pax1 homologs (Pax1a and Pax1b) in zebrafish are also expressed in the developing osteogenesis and endodermal pharyngeal sacs [15].Pax9 is one of the best-characterized transcription factors involved in human tooth development, capable of influencing the number, position, and morphology of an individual tooth.Mutations in the Pax9 gene have been reported to be associated with various types of dental hypoplasia and other inherited dental defects or variants [16].The Pax1/9 gene in B. taurus is presumed to be functionally similar to the Pax1/9 gene in buffalo and the Zebu.The expression of the paired-box genes Pax1 and Pax9 is associated with limb skeleton development [4].We speculate that Pax1/9 affects the growth traits of B. taurus.
Pax2 is essential for the development of the genitourinary system, neural tube, optic vesicles, optic cup, and optic tract [17], and Pax2 mutations could cause eye defects [18].The Pax2 gene has significant genetic effects on disease resistance in buffalo and the milk fatty acids of dairy cattle [19,20].Pax5 is a key transcription factor that determines β-cell stereotypy and development.Pax5 represses genes inappropriate for the β-cell lineage and induces gene expression required for β-cell development.Moreover, Pax5 posttranscriptionally downregulates the expression of phosphatase and tensin homolog (PTEN) to promote the differentiation and survival of mature β-cells, thereby promoting humoral immunity [21,22].Pax5 affects coat color, which might drive the differences among Chinese yellowish coated breeds and those in the greater Far East region [23,24].Pax8 belongs to a class of spectrum survival genes that are required for both the normal development of certain tissues and the proliferation of cancer cells [24,25].Pax2 and 8 are over-represented in biological processes related to kidney organogenesis in beef-cattle placental tissues [26].The Pax2/5/8 subfamily affects coat color and reproductive and milk performance in cattle, but there is no clear understanding of the B. taurus meat traits.
The transcription factors encoded by Pax3 and Pax7 are among the first to be expressed in the embryo, and both are key regulators of myogenesis capable of influencing the development of mammalian limb and most hypothenar muscles [27].Pax3-positive muscle stem cells become sensitive to environmental stress when Pax3 function is impaired, and the Pax3-mediated induction of mammalian targets of rapamycin complex 1 (mTORC1) is required for protection [28].Pax3 could regulate the neural crest and the onset of myogenic differentiation in the somites, both of which represent the synchronous development of the neural crest and skeletal muscle lineages [29,30].Pax7 plays key roles from early central nervous system development to brain maturation and drives the formation of the vertebrate visual system by determining the formation and detection of the superior colliculus [31].Pax3 plays an important role in early embryonic skeletal muscle formation and can regulate the myogenic-determinant myogenic factor 5 (Myf5) and myogenic differentiation (MyoD).Pax7 plays a dominant role in adult growth and muscle regeneration, and Pax7 deficiency results in satellite cell deficiency, muscle atrophy, and early postnatal mortality in mice [32,33].The polymorphism of Pax3 affects the growth traits of Chinese domestic cattle [12].In addition, B. taurus Pax7 is closely related to buffalo Pax7 and Zebu Pax7.The Pax3/7 gene is conserved during the evolution of B. taurus and may be a key gene in regulating bovine growth.
Pax4 is a direct target of neurogenin 3, which regulates β-cell specificity and is a key gene in pancreatic development.Mice lacking Pax4 develop severe hyperglycaemia and die within days of birth due to a lack of mature pancreatic cells [34].Pax6 is a major transcription factor in early eye development and is extremely important in the postnatal development of the eye [35].Human Pax6 shares 90% and 96% sequence homology with Drosophila and zebrafish, respectively.During eye development, two structurally similar Pax6 isoforms perform two distinct functions by activating or repressing different target genes, and both the knockout and overexpression of Pax6 inhibit normal eye development [36].Pax4 regulates bovine adipocyte differentiation and lipid homeostasis and Pax6 is an important transcription factor affecting the milk fat traits of dairy cattle [37,38].Pax4 affects tropical adaption between Indian Zebu cattle and riverine buffalo [39].

Conclusions
In this study, nine Pax genes were identified from the genome of B. taurus, and these could be divided into four subfamilies: Pax1/9, Pax2/5/8, Pax3/7, and Pax4/6.The Pax gene family members were located in the nucleus.Molecular phylogenetic analysis showed that the structures and sequences of Pax genes in buffalo, the Zebu, and B. taurus were similar.The species and quantity distributions of the domains and motifs of the subfamily members were consistent.In addition, evolutionary analysis revealed that the Pax domain is highly conserved in all Pax gene family members.Protein interaction network analysis showed that only the Pax3 and Pax7 genes had direct interaction among the Pax genes.We believe that Pax1/9 has an effect on the growth traits of buffalo and domestic cattle.The Pax3/7 gene is conserved in the evolution of buffalo and domestic animals and may be a key gene regulating growth in B. taurus.The Pax2/5/8 subfamily affects coat color, reproductive performance, and milk production performance in cattle.The Pax4/6 subfamily has an effect on the milk fat percentage of B. taurus.In future studies, the role of the Pax gene family on B. taurus needs to be verified in bovine myoblasts or in vivo.This study's results will provide guidance for further marker-assisted selection breeding tar-geting the Pax gene family to improve B. taurus' yield performance.

2. 4 .
Physicochemical Properties and Subcellular Localization of Pax Gene Family in B. taurus In order to explore the physicochemical properties of Pax gene family in domestic cattle, we uploaded Pax sequences to ExPasy (https://web.expasy.org/compute_pi/)(assessed on 20 May 2024) and obtained the length, molecular weight (MW), and isoelectric point (PI) data of Pax gene.In order to determine the location of Pax gene family members in cells, we uploaded the amino acid sequences of Pax gene family members to the WoLF PSORT (https:// wolfpsort.hgc.jp/)(assessed on 20 May 2024) website and selected animal protein species to analyze and predict the subcellular localization of Pax gene family members.

2. 5 .
Co-Linearity Analysis and Chromosome Localization of Pax Gene in B. taurus In order to determine the specific location of each Pax gene family member, the genome annotation files (gff) and gene list files of B. taurus, B. indicus, and B. bubalis downloaded from NCBI were inputted into MCScanX to achieve chromosomal localization of the Pax gene family.In order to explore the link between Pax gene family members of B. taurus, B. indicus, and B. bubalis, we imported genome files and annotation files of B. taurus, B. indicus, and B. bubalis into TBtools-II v2.102 and analyzed the data through One-Step MCScanX plug-in of the software.The Dual Systeny Plot for MCScanX plug-in was used to visualize the results to obtain the co-linearity maps of Pax genes of B. taurus, B. indicus, and B. bubalis.2.6.Multiple-Sequence Alignment of Pax Gene in B. taurus

Figure 1 .
Figure 1.NJ evolutionary tree of Pax gene family in domestic cattle, Zebu, buffalo, sheep, and pig.

Figure 1 .
Figure 1.NJ evolutionary tree of Pax gene family in domestic cattle, Zebu, buffalo, sheep, and pig.

Figure 2 .
Figure 2. Gene structures of B. taurus Pax gene family.

3. 4 .
Co-linearity Analysis and Chromosome Localization of Pax Gene in B. taurus Using MCScanX, we located members of the Pax gene family in B. taurus, B. indicus, and B. bubalis.The analysis results (Figure 4) indicate a highly consistent distribution of Pax genes between B. taurus and B. indicus.This suggests a potentially high functional similarity between the Pax gene families in B. taurus and B. indicus, implying that studying the Pax gene family in B. indicus could help in assessing the functionality of the Pax gene family in B. taurus.Co-linearity analysis provides valuable insights into evolutionary relationships and polyploid events.According to the previous evolutionary tree of the Pax gene family, B. taurus, B. bubalis, and B. indicus exhibit a closer genetic affinity.Therefore, through the co-linearity analysis of these three cattle species and considering the known functions of Pax genes in B. bubalis and B. indicus, we aimed to explore the functions of B. taurus Pax genes that exhibit co-linearity.Using TBtools, we generated graphical representations of the co-linearity analysis results for these species.B. bubalis shows co-linearity with B. taurus and B. indicus Pax genes, displaying an overall one-to-one correspondence across different species (Figure 5).Among them, pax gene family members in B. taurus and B. indicus also exist on the same chromosome, which indicates that Pax gene differentiation in B. taurus and B. indicus has not gone far.This suggests potential functional similarity between Pax gene family members in B. taurus and Pax gene family members in B. indicus.The Pax gene function of B. taurus could be evaluated based on the Pax gene function in B. indicus and B. bubalis.

3. 4 .
Co-linearity Analysis and Chromosome Localization of Pax Gene in B. taurus Using MCScanX, we located members of the Pax gene family in B. taurus, B. indicus, and B. bubalis.The analysis results (Figure 4) indicate a highly consistent distribution of Pax genes between B. taurus and B. indicus.This suggests a potentially high functional similarity between the Pax gene families in B. taurus and B. indicus, implying that studying the Pax gene family in B. indicus could help in assessing the functionality of the Pax gene family in B. taurus.

Figure 4 .
Figure 4. Chromosomal assignment of the Pax gene family.Co-linearity analysis provides valuable insights into evolutionary relationships and polyploid events.According to the previous evolutionary tree of the Pax gene family, B.

Figure 4 .
Figure 4. Chromosomal assignment of the Pax gene family.

Figure 5 .
Figure 5. Co-linearity analysis of the Pax gene family in B. taurus, B. indicus, and B. bubalis.

Figure 5 .
Figure 5. Co-linearity analysis of the Pax gene family in B. taurus, B. indicus, and B. bubalis.

Genes 2024 , 15 Figure 6 .
Figure 6.Multiple-sequence alignment of the Pax gene family in B. taurus.

Figure 6 .
Figure 6.Multiple-sequence alignment of the Pax gene family in B. taurus.

Figure 7 .
Figure 7. Three-dimensional structures of Pax gene family proteins.

Figure 7 .
Figure 7. Three-dimensional structures of Pax gene family proteins.

Figure 8 .
Figure 8.Protein interaction network of the Pax gene family.

Figure 8 .
Figure 8.Protein interaction network of the Pax gene family.

Table 1 .
Differentially conserved motifs in B. taurus Pax gene family.Physical and Chemical Properties and Subcellular Localization of Pax Gene Family in B. taurus ExPasy was used to analyze the lengths, molecular weights (MWs), and isoelectric points (PIs) of the Pax gene family.The results showed that the lengths of the Pax gene family ranged from 328 to 505 bp and the molecular weights ranged from 35,573.8 to 55,100.5 Da.The isoelectric points (PIs) ranged from 7.93 to 10.61, all of which are alkaline (Table2).

Table 2 .
Physicochemical properties and subcellular localization of the Pax gene family in B. taurus.